IDENTIFICATION OF AN INSULIN-RESPONSE ELEMENT IN THE FATTY-ACID SYNTHASE PROMOTER

We have previously reported that insulin increases fatty acid synthase (FAS) gene transcription, and that sequences responsible for positive regulation are located within the first 332 base pairs of the FAS pro moter. To define minimal sequences required for insulin regulation wit hin this region, chimeric constructs containing serial 5' deletions st arting at -318 and extending through position +67 of the rat FAS gene ligated to the luciferase reporter gene were transfected into 3T3-L1 a dipocytes. Insulin treatment at 10 nm increased luciferase activity 2- 3-fold in 3T3-L1 adipocytes transfected with constructs containing pro gressive deletions from -318 to -67. This stimulation of the FAS promo ter activity by insulin was dose-dependent. However, no effect of insu lin was observed when fusion constructs containing FAS promoter sequen ces spanning from -25 or from -19 to +67 were transfected into adipocy tes. These results suggest that the insulin response sequences of the FAS gene may be located in the region from -67 to -25. DNase I footpri nting using liver nuclear extracts revealed a protected region spannin g -71 and -50 in addition to a region near the putative TATA box. Gel mobility shift assays using the sequence from -71 to -50 as a probe re vealed nuclear factor(s) from mouse liver and 3T3-L1 adipocytes that s pecifically complexed with this sequence. Mutational analysis of this region showed that sequences between -68 and -60 are essential for rec ognition and interaction with a trans-acting factor(s). Moreover, when three tandem repeats of the sequences spanning -68 to -52 were linked to the SV40 promoter and used for transfection, luciferase activity i ncreased 3.6-fold in response to insulin treatment. Thus, we have iden tified novel cis-acting DNA sequences responsible for insulin regulati on of the FAS gene, which interact with nuclear protein(s) from liver and adipocytes and which are found to share limited homology to insuli n response sequences present in other genes.